Transposition assembly for gene transfer in eukaryotes

ABSTRACT

A transposition assembly for the transfer of a DNA fragment of interest into the ribosomal nuclear DNA of an eukaryotic cell. An insertion means, an eukaryotic cell and a pharmaceutical composition comprising said transposition assembly, as well as a method for the in vitro transfer of said DNA fragment, are also disclosed.

The present invention relates to a transposition assembly allowing thetransfer of genes of interest into the genome of a cell or of aeukaryotic organism. Such an assembly is particularly useful for genetherapy purposes.

Numerous elements which can be employed by the integration of genes ofinterest into the eukaryotic genome have been described in the prior artpublications. The conventional elements are either integrative vectors,for example retroviral vectors, or nonintegrative vectors, for exampleadenoviral vectors. However, these prior art vectors not only haveadvantages.

In fact, the retroviral vectors are integrated randomly into thecellular genome in a nonspecific manner. Thus the risk of insertionalmutagenesis linked either to the inactivation of genes essential to thecell, or to the activation of oncogenes, which can give rise to atumoral proliferation, is not to be neglected before contemplating theiruse in human gene therapy.

As far as the nonintegrative vectors are concerned, they havedisadvantages linked to an instability on account of theirnonintegration into the cellular genome, which necessitates theirregularly repeated administration. Within the context of a gene therapyintended for man, in the long term this risks posing problems ofimmunization against the recombinant virus in regularly treatedpatients.

A third type of method, described more recently, allows the transfer ofgenes of interest by homologous recombination into a defined site of thecellular genome. However, the technique of homologous recombinationagain comes up against numerous technical difficulties. In addition,nonhomologous recombination events can equally be produced, so that therisk of insertional mutagenesis remains.

Moreover, the prior art has for a long time established the scheme oforganization and of expression of ribosomal DNA (rDNA) in eukaryotes(Reeder, Trends Genet. 1990, 6, 390-395). Generally speaking, rDNA isformed by multiple copies of transcription units arranged in pairs. Atranscriptional unit is composed of genes coding respectively for the18S or 16S, 5-8S and 28S or 26S rRNAs, which enter into the constitutionof the ribosome (below designated 18S rRNA gene etc.). Each gene isseparated from the following by transcribed sequences whose exact roleis still not defined. The three rRNA genes contained in atranscriptional unit are placed under the control of a unique promotersituated upstream in the nontranscribed region which separates one unitfrom the following. The units of rRNA are transcribed by the RNApolymerase I in a long molecule of precursor rRNA (pre-rRNA), which isthen matured to produce the three principle types of rRNA associated tothe ribosomal sub-units.

Numerous publications have reported the frequent presence of foreigngenetic elements in the rDNA of eukaryotes. Among these geneticelements, introns of class I or II and retroposons, for example of classR1, R2 or R3, can be mentioned. The presence of these foreign elementscan possibly inactivate a fraction of the transcriptional units of therDNA. This does not seem to have serious consequences for the life ofthe organisms containing them. This phenomenon has particularly beendescribed in the drosophila.

The introns of classes I and II are defined by the existence ofpreserved sequences forming structural elements characteristic of eachof the classes such as defined by Cech and Bass (1986, Annu. Rev.Biochem 55, 599-629). Several authors have observed that certain intronsof classes I and II are mobile. They can be copied and transferredspecifically into copies of genes which are devoid of them (intron⁻).This transposition process, at least in the majority of cases, turns outto be specific from the point of view of the insertion region.

Among the sixty or so introns of class I characterized until now, themajority are localized in the genes of the mitochondria and of thechloroplasts. The prior art mentions, in three cases only, the presenceof mobile introns of class I in the nuclear genes. These introns havebeen demonstrated at the level of the rRNA genes of three species oflower eukaryotes, respectively the 26S rRNA genes of several strains ofTetrahymena (Kan et Gall, 1982, Nucl. Acids Res., 10, 2809-2821) and ofthe Carolina strain of Physarum polycephalum, (Muscarella et Vogt, 1989,Cell, 56, 443-454; this mobile intron being designated intron 3 below)and finally in the 16S rRNA gene of Pneumocystis carinii (Edman et al.,1988, Nature 334, 519-522). Until now, it has not been possible todemonstrate the presence of introns interrupting the rRNA genes ofhigher eukaryotes.

The intron 3 of Physarum polycephalum is the best characterized. Itsmobility has been demonstrated experimentally (Muscarella et Vogt, 1989,Cell, 56, 443-454). This intron codes in part for a protein of 160 aminoacids (Muscarella et al., 1990, Mol. Cel. Biol., 10, 3386-3396). Theinitiation codon of the putative translation is situated upstream of theintron, at the 3′ end of the adjacent exon sequence at the 5′ end of theintron. The protein encoded by the intron 3 is an endonuclease whichrecognizes a target sequence of at least 18 base pairs (bp) present inthe insertion region and is capable of cleaving this sequence exactly atthe level of the site of insertion of the intron 3 (Muscarella et Vogt,1989, Cell, 56, 443-454).

The target sequence recognized by the endonuclease comprises thefollowing sequence: 5′ CTATGACTCTCTTAAGGTAGCCAAA3′ (SEQ ID NO:1). It isassumed that the transposition of the intron 3 is initiated by therecognition of the particular target sequence by the endonucleasespecifically encoded by the intron 3, followed by a cutting of the twostrands of the DNA molecule at the level of this particular sequence,thus liberating the insertion site. Then, by an exchange of fibersinvolving the flanking sequences at the 5′ and 3′ ends of the insertionsite, copying of the intron sequence and recombination, the intron 3 isinserted into the copy of the intron⁻ rRNA gene in a precise andsite-specific manner. Once the intron 3 is inserted into a copy of theintron⁻ gene, the target sequence recognized by the endonuclease isinterrupted by the intron sequence in the following manner: 5′CTATGACTCTCT (SEQ ID NO:2) intron 3 TAAGGTAGCCAAA3′ (SEQ ID NO:3).

On the other hand, retroposons equally seem to be present in the rDNA ofcertain eukaryotes. Generally speaking the retroposons form a very hugeand very heterogeneous group, especially at the level of theirnucleotide sequence. Retroposon is understood as meaning elementsrelated to the retroviruses, but devoid of long terminal repeats (LTR).They comprise one or more open reading frames capable of coding forproteins having a homology with retroviral proteins, such as, forexample, reverse transcriptase (Jakubczak et al., 1991, Proc. Natl.Acad. Sci. USA, 88, 3295-3299).

In a certain number of nonmammalian eukaryotes, especially insects,retroposons having a remarkable specificity of insertion have beenfound, localized at the level of the rRNA genes. The mobility of certainof these has been observed. Several families (especially R1, R2 and R3)have been defined as a function of their specific insertion region intothe rRNA genes.

The retroposons of the classes R1 and R2 are especially illustrated bythe retroposons R1Bm and R2Bm of Bombyx mori (Xiong and Eickbush, 1988,Cell, 55, 235-246; Xiong and Eickbush, 1988, Mol. Cell. Biol., 8,114-123; Xiong et al., 1988, Nucl. Ac. Res., 16, 10561-10573) and theretroposons R1Dm and R2Dm of Drosophila melanogaster (Jakubczak et al.,1990, J. Mol. Biol., 212, 37-52). They contain air open reading framecapable of coding for a protein of great size whose central part has ahomology with the reverse transcriptase family. Xiong and Eickbush(1988, Cell, 55, 235-246) report that the protein encoded by theretroposon R2Bm of Bombyx mori moreover has an endonuclease activity.This nuclease recognizes and cleaves a target sequence contained in theinsertion region of R2Bm situated in the 28S rRNA gene of the insectgenome.

A novel class of ribosomal retroposons, designated R3, has recently beendemonstrated. Protein encoded by this element has still not beencharacterized. However, the specific insertion region of the element R3into the rDNA of Scaria coprophila has been described (Kerrebrock etal., J. Mol. Biol., 1989, 210, 1-13).

Beside retroposons, there are a large number of mobile genetic elementswhich have sometimes been designated under the name of retroposons bythe authors describing them, but of which it has still not been shownthat they code for a protein having a homology with the family ofretroviral proteins, especially reverse transcriptase. This type ofelements has equally been found at the level of well-defined sequencesof rRNA genes of organisms containing them (see, for example, Back etal., 1984, EMBO J., 3, 2523-2529).

Surprisingly, it has now been found that on the one hand the targetsequences recognized by the endonucleases encoded by the two mobilegenetic elements for which the data are available (intron 3 of Physarumpolycephalum and retroposon R2Bm of Bombyx mori) and on the other handthe insertion regions of certain mobile genetic elements of which it hasstill not been shown that they code for an endonuclease and which havebeen disclosed in the prior art (Jakubczak et al., 1991, Proc. Natl.Acad. Sci. USA, 88, 3295-3299; Paskwitz et Collins, 1989, Nucleic AcidRes. 17, 8125-8133; Kerrebrock et al., 1989, J. Mol. Biol., 210, 1-13;Kan and Gall, 1982, Nucleic Acid Res., 10, 2809-2821; Edman et al.,1988, Nature, 263, 519-522), are conserved in the rRNA genes of variousmammals, especially man and the mouse. Thus, the sequences of insertionregions of mobile introns of Tetrahymena, of Pneumocystis carinii and ofPhysarum polycephalum, and of R2Bm retroposons of Bombyx mori, and R3 ofScaria coprophila are conserved identically in the rRNA genes ofmammals. On the other hand, the sequences of the insertion region of theretroposons R1Dm and R1Bm are homologous but nonidentical to a sequencepresent in the mammalian 28S rRNA gene.

Consequently, the present invention relates to a transposition assemblyintended for the transfer of a DNA fragment of interest into the genomeof a eukaryotic host cell, which comprises an integration cassette,essentially formed by a mobile genetic element in the midst of which isinserted the said DNA fragment of interest; the said integrationcassette being capable of being integrated in a site-specific fashioninto a specific insertion site situated in the ribosomal nuclear DNA ofthe said host cell.

A transposition assembly according to the invention has the advantage ofallowing the integration of a DNA fragment of interest into a definedand nonessential region of the cellular genome. Such an assembly isespecially useful for gene therapy purposes.

For the purposes of the present invention, the integration cassette mustcomprise a mobile genetic element which has the capacity for integratingitself by a site-specific mechanism into the nuclear rDNA, in particularat the level of the 28S, 18S or 5-8S rRNA gene. More precisely, thismobile genetic element must integrate itself into an insertion sitesituated in an insertion region of which the sequence is conserved in anidentical or nonidentical manner in the nuclear rDNA of the host cell.

For a better understanding, it is specified that the term “insertionsite” defines the place between 2 nucleotides where a mobile geneticelement is inserted. In the same way, “insertion region” is understoodas meaning the nucleotide sequences at the 5′ and 3′ end of theinsertion site which are required for the site-specific transposition.Generally speaking, each mobile genetic element possesses an insertionregion which is specific to it. As mentioned above for the retroposonsR1Bm and R1dm, it is not required that the insertion region in the hostcell comprises in an identical manner the sequences of the naturalinsertion region (in the organism of origin). Thus, the sequence of theinsertion region of the said mobile genetic element in the eukaryotichost cell will present a degree of homology with the sequence of thenatural insertion region greater than 80%, advantageously greater than90% and preferably greater than 95%.

The mobile genetic element is advantageously selected from amongstmobile introns of class I or II, and retroposons such as the retroposonsof class R1R2 [sic] or R3.

A genetic mobile element can be formed by a functional fragment or avariant of the said element. Functional fragment is understood asmeaning any fragment which has conserved the capacity of beingintegrated of the complete element. A variant can comprise one or moremutations with respect to the natural nucleotide sequence of theelement, especially the substitution, addition or deletion of one ormore nucleotides, on condition that these mutations do not alter thefunction.

More particularly, the mobile genetic element is selected from amongst:

the intron 3 of Physarum polycephalum, Carolina strain, a fragment or avariant of the said intron,

the mobile intron of class I of Tetrahymena, a fragment or a variant ofthe said intron,

the mobile intron of class I of Pneumocystis carinii, a fragment or avariant of the said intron,

the retroposon of R2Bm of Bombyx mori, a fragment or a variant of thesaid retroposon and,

the retroposon R3 of Scaria coprophila, a fragment or a variant of thesaid retroposon.

The presence in this mobile genetic element of all or part of an openreading frame capable of coding for an integrase is a preferredcharacteristic.

Generally speaking, integrase is understood as meaning a protein havingan enzymatic activity allowing it to participate directly or indirectlyin the trans-position of the mobile genetic element specifically at thelevel of its insertion site in the nuclear rDNA of a eukaryotic cell.

Advantageously, the integrase can be especially formed by:

(1) a protein having an endonuclease activity capable of recognizing aspecific target sequence included in the insertion region of the mobilegenetic element which codes for it and of cleaving the said targetsequence, or

(2) a protein having a homology with the family of reversetranscriptases.

It is possible to mention, for example, the endonuclease encoded in partby the intron 3 of Physarum popycephalum. This endonuclease initiatesthe transposition of the intron 3 on recognizing its target sequence andcleaving the DNA molecule at the level of this specific sequence.

It is equally possible to mention the protein encoded by the retroposonR2Bm of Bombyx mori. The endonuclease activity of the said protein isprobably involved in the transposition of R2Bm at the level of itsspecific insertion region, but by a mechanism which is not known todate.

The DNA fragment of interest can be introduced into the mobile geneticelement by the conventional techniques of genetic engineering. It can beintegrated in or outside of the open reading frame coding for a possibleintegrase.

According to a particularly advantageous aspect of the invention, theDNA fragment of interest is inserted in the open reading frame in orderto prevent the expression of the integrase. This introduces a securityfeature to avoid the uncontrolled propagation of the integrationcassette in the genome of the host cell.

In this case, the specific integrase of the mobile genetic elementemployed in the transposition assembly according to the invention shouldbe supplied to the host cell in a transitory manner during a time whichis sufficiently long to allow the transposition of the integrationcassette into its specific insertion region. The means of supplying aprotein in trans to a eukaryotic cell are numerous and known to theperson skilled in the art.

For example, the transposition assembly according to the invention canbe introduced into a vector (such as defined below), moreover comprisingan expression cassette of the specific integrase of the mobile geneticelement present in the transposition assembly.

In an alternative manner, the eukaryotic host cell can be transfected inparallel with a helper vector comprising an expression cassette of theintegrase or the integrase can be supplied in purified form to the hostcell.

The DNA fragment of interest can be any fragment capable of beingtranscribed to RNA to produce an anti-sense RNA for example acomplementary RNA sequence of a pathogenic gene capable of forming aduplex with a pathogenic transcript in order to inhibit the translationto pathogenic protein. A pathogenic gene is:

(1) a gene which is not present in the eukaryotic cells, for example agene present in the genome of a pathogenic organism (bacteria, virus orparasite), or

(2) a homologous gene or a mutated homologous gene, for example anoncogene, which is present but normally not expressed in the normaleukaryotic cells and whose abnormally induced expression can cause adisorder such as cancer.

In an alternative fashion, the DNA fragment of interest can code for aprotein of interest and, in a preferred manner, a protein whose absenceof expression or expression in abnormal quantity or in mutant form isassociated with a genetic disorder.

The DNA fragment of interest can code for a mature protein or aprecursor of this. In the first case, it comprises the sequence codingfor a mature protein allowing the expression of the protein inintracellular fashion. In the last case, the DNA fragment of interestcan equally include a signal sequence allowing the secretion of the saidprotein of the host cell. The DNA fragment of interest can code for achimeric protein arising from the fusion of various sequences of origin.

The examples of proteins which can be encoded by the DNA fragment ofinterest comprise:

cytokines, such as alpha interferon, gamma interferon and differenttypes of growth factors,

membrane receptors, such as the receptors involved in the transmissionof signals from the surface to the interior of cells and the receptorsrecognized by pathogenic organisms, such as the CD4 receptor present atthe surface of T lymphocytes and recognized by the envelopeglyco-protein of the HIV virus (Human Immunodeficiency Virus),

enzymes, such as the ribonucleases and the thymidine kinase (TK) of thetype I herpes simplex virus (HSV-1). The latter has a superior affinitywith respect to the mammalian cell TK enzyme for certain analogs ofnucleosides, such as acyclovir or gancyclovir. The enzyme TK-HSV-1converts the analogs into precursors of nucleotides. These toxicprecursors are then incorporated into the DNA of cells in a state ofreplication. This incorporation allows the cells in division, such ascancer cells, to be killed specifically,

inhibitors of enzymatic activity, such as alpha 1 antitrypsin,antithrombin III, protein C and specific protease inhibitors of apathogenic organism,

coagulation factors, such as factor VIII, factor IX and thrombin,

proteins involved in ionic channels, such as the protein CFTR (CysticFibrosis Transmembrane Conductance Regulator),

proteins capable of inhibiting the activity of a protein produced by apathogenic gene, such as the suppressor antigen of tumor p53,

variants of pathogenic proteins mutated so as to alter their biologicalfunction, such as trans-dominant mutants of the regulator protein TAT ofthe HIV virus capable of competing with the native viral protein forlinkage to the target sequence, preventing activation of the expressionof the viral genes and,

antigenic epitopes allowing immunity of the host cell to be increased.

The DNA fragment of interest can be mutated so as to allow theexpression of a protein of interest whose biological properties aremodified, for example a variant of alpha 1 antitrypsin whose methionineresidue in position 358 of the active site has been replaced by aleucine. Such a variant is functional under oxidation conditions such asinflammation conditions.

Once the integration cassette is introduced into the rDNA of the hostcell, the DNA fragment of interest can be placed under the dependence ofthe promoter of the transcriptional unit of the rDNA of the host cell.Alternatively, the DNA fragment of interest can be placed under thecontrol of appropriate expression elements inserted into the mobilegenetic element. Expression here signifies transcription to RNA and/ortranslation of this RNA to protein.

According to this alternative, the control elements of the expressionespecially comprise an appropriate promoter. Such promoters are wellknown to the person skilled in the art and are inserted upstream of theDNA fragment of interest by the conventional techniques of geneticengineering.

The promoter retained can be a promoter recognized by RNA polymerase I,so as to favor the expression of the DNA fragment of interest, afterintegration of the integration cassette into the rRNA genes of theeukaryotic host cell. For example, the DNA fragment of interest can beplaced under the control of regulation elements included in thenontranscribed regions of the rDNA involved in the transcription of therRNA genes. Such promoters will be chosen so as to be functional in theeukaryotic host cell which has been retained.

In an alternative manner, the promoter retained can be a promoterrecognized by RNA polymerase II. Such promoters are well known to aperson skilled in the art. The promoter can be isolated from a cellulargene or from a virus. It can be ubiquitous, allowing a permanentexpression of the DNA fragment of interest in all the types of hostcells. The term promoter equally includes a regulatable promoter, forexample a tissue-specific promoter. It is possible especially to mentionthe promoter of the HMG gene (hydroxymethylglutaryl coenzyme Areductase), of the TK gene of the HSV-1 virus, of the SV40 virus (Simianvirus 40), the promoters EIII and MLP (Major Late Promoter) of theadenovirus, the LTR of the MoMuLV virus (Moloney Murine Leukemia Virus),the promoter of the FIX gene which confers a liver-specific expressionor the promoter of specific immunoglobulin genes of lymphocyte cells.

In an advantageous manner, the transposition assembly moreover comprisesan integration cassette of elements allowing or favoring thesite-specific integration of this cassette. According to a particularmode of the invention, especially when the mobile genetic elementemployed is a mobile intron of class I or II, the transposition assemblyaccording to the invention moreover comprises:

at the 5′ end of the integration cassette a region of at most 10 kb,having at least 80% homology with the sequences of the rRNA gene of thehost cell immediately adjacent at the 5′ end at the said insertion site;and

at the 3′ end of the integration cassette a region of at most 10 kbhaving at least 80% homology with the sequences of the rRNA gene of thehost cell immediately adjacent at the 3′ end at the said insertion site.In fact, it can be advantageous to place the said integration cassettein an rDNA environment, particularly when the transposition involves aphenomenon of exchange of strands as has been shown especially for theintron 3. The exchange of strands involves the intron+donor sequences(of the transposition assembly) and the intron-recipient sequences (ofthe host cell). Only the integration cassette will be transposed intothe genome of the host cell. The sequences of the rRNA gene immediatelyadjacent at the 5′ and 3′ ends of the insertion site intervene uniquelyin the transposition process.

In order to favor the transposition of the integration cassette, it willbe preferred to have perfect homology between the donor and recipientsequences involved in the strand exchange. Thus, a preferredtransposition assembly will moreover comprise:

at the 5′ end of the integration cassette a region of at most 10 kb,having 100% homology with the sequences of the rRNA gene of the hostcell immediately adjacent at the 5′ end at the said insertion site; and

at the 3′ end of the integration cassette a region of at most 10 kb,having at least 100% homology with the sequences of the rRNA gene of thehost cell immediately adjacent at the 3′ end at the said insertion site.

The sequences of the rRNA gene immediately adjacent at the 5′ and 3′ends at the insertion site can be isolated according to the classicaltechniques of genetic engineering, for example by cloning or PCR(Polymerase Chain Reaction) or chemical synthesis. More particularly,these sequences will have a length of at most 10 kb, advantageously atmost 3 kb and preferably at most 0.4 kb.

The present invention equally relates to a means of introduction of atransposition assembly according to the invention into a eukaryoticcell. Such means consisting in introducing a nucleic acid into aeukaryotic cell are generally known to a person skilled in the are.

For the purposes of the present invention, the transposition assemblyaccording to the invention can be introduced in a means of introductionselected from amongst the delivery vehicles of the liposome andsynthetic cationic lipid type and the cloning and expression vectorsclassically utilized in the eukaryotic cells. Encapsulation in adelivery vehicle and the techniques of cloning in vectors are classicaltechniques known to the person skilled in the art. However, otherprotocols allowing a nucleic acid to be introduced into a cell canequally be employed, such as, for example, calcium phosphateprecipitation, the DEAE-dextran technique, direct injection of thenucleic acid into a cell or the bombardment of gold microparticlescovered with nucleic acid in the cells of an animal. The nucleic acidcan be introduced in supercoiled, circular or linear form.

Advantageously, the means of introduction according to the invention isa vector comprising the elements appropriate for its maintenance in thehost cell for a time which is sufficiently long to allow the transfer ofthe integration cassette into the insertion region. Such a vector has tobe capable of entering a higher eukaryotic cell, of remaining,preferably, in extra-chromosomal form and of being maintained in thecell for a time which is sufficiently long to allow the transposition ofthe integration cassette into the genome of the host cell. Such a vectorcan be in the form of a plasmid or of a viral vector.

Preferably, the means of introduction according to the invention is avector of the nonintegrative type. It is possible to mention a vectorderived from the herpes simplex virus or from an adenovirus.Particularly preferably, the means of introduction according to theinvention is a vector derived from an adenovirus, such as, for example,type 5 adenovirus.

According to an advantageous mode and as recalled above, the means ofintroduction according to the invention can moreover contain anexpression cassette allowing the production of the specific integrase ofthe transposition assembly.

Such an expression cassette comprises the DNA fragment coding for thesaid integrase, placed under the control of appropriate elementsallowing its expression. Appropriate elements allowing its expression isunderstood as meaning the whole of the elements allowing thetranscription of the said fragment of DNA to mRNA and the translation ofthe mRNA to protein. These elements especially comprise an appropriatepromoter, preferably a promoter recognized by the RNA polymerase II andallowing a strong and ubiquitous expression, for example the promoterSV40.

The means of introduction according to the invention can moreovercomprise an expression cassette allowing the expression of a selectiongene allowing the detection and isolation of the host cells comprisingthe said means of introduction. In the context of the invention, thegene coding for the selection marker can be under the control ofappropriate elements allowing its expression in the host cell, such asdefined above.

The invention is equally related to an in vitro transfer process of aDNA fragment of interest into the genome of a eukaryotic host cell atthe level of the ribosomal nuclear DNA, according to which atransposition assembly according to the invention or a means ofintroduction according to the invention is introduced into the said hostcell.

Advantageously, it is moreover possible to supply the specific integraseof the transposition assembly according to the invention to the hostcell in the in vitro process according to the invention.

The invention equally relates to a eukaryotic cell comprising atransposition assembly according to the invention or a means ofintroduction according to the invention. The said cell willadvantageously be a mammalian cell, and preferably a human cell.

The present invention equally relates to a pharmaceutical compositioncomprising by way of therapeutic agent, a transposition assemblyaccording to the invention, a means of introduction according to theinvention or a cell according to the invention, in association with acarrier which is acceptable from a pharmaceutical point of view.

The pharmaceutical composition according to the invention isparticularly intended for the preventive or curative treatment ofdisorders such as:

genetic disorders, such as, for example, hemophilia or mucoviscidosis,

cancers, such as, for example, those induced by oncogenes or viruses,

retroviral disorders, such as, for example, AIDS (AcquiredImmunodeficiency Syndrome), and

recurrent viral disorders, such as, for example, viral infections causedby the herpes virus.

A pharmaceutical composition according to the invention can bemanufactured in a conventional manner. In particular, a therapeuticallyefficacious quantity of a therapeutic agent is united with a supportsuch as a diluent. A composition according to the invention can beadministered by any conventional route in use in the field of the art,in particular by the subcutaneous route, by the intramuscular route, bythe intravenous route or by the intratracheal route. Administration cantake place in a single dose or repeated one or more times after acertain time interval. The route of administration and the appropriatedose vary as a function of various parameters, for example of theindividual treated or of the DNA fragment of interest. A pharmaceuticalcomposition can moreover comprise an adjuvant acceptable from apharmaceutical point of view.

Advantageously, the pharmaceutical composition according to theinvention moreover comprises an integrase or an integrase expressioncassette.

The invention equally extends to a method of treatment according towhich a therapeutically efficacious quantity of a transposition assemblyaccording to the invention, a means of introduction according to theinvention or a cell according to the invention is administered to apatient having need of such a treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated below by reference to FIG. 1 whichschematically represents a transposition assembly (1) (large blank box)which comprises from 5′ to 3′: the sequences of the rRNA gene (6)(hatched box) at the 5′ end of the insertion site (5) (black lines), anintegration cassette (3) (small blank box) formed by a mobile geneticelement (4) (horizontally ruled box) in the midst of which is inserted afragment of DNA of interest (2) (stippled box) and the sequences of therRNA gene (7) (hatched box) at the 3′ end of the insertion site (5).

EXAMPLES Example 1 Construction of a Transposition Assembly forSite-specific Integration into the 28S rRNA Genes of a Human Cell,Employing Intron 3 of Physarum polycephalum Carolina Strain

The example refers to a transposition assembly which comprises

(1) the intron 3 modified so as to create a unique site of restrictionXhoI in the intron sequence. The site XhoI will allow a fragment of DNAof any interest whatsoever to be introduced. In fact, the DNA fragmentof interest can be isolated so as to have sticky ends compatible withthe ends generated by an XhoI digestion (restriction fragment XhoI orSailI or XhoI-SalI or addition of XhoI and/or SailI linkers ormutagenesis directed in order to create the required sites XhoI and/orSalI). Alternatively, the cloning site XhoI can be treated, for example,with Mung bean nuclease in order to generate free ends between which itis possible to clone any fragment of DNA of interest with free ends. TheDNA fragment of 0.94 kb comprising the intron 3 can be isolated,especially by cloning or PCR, or synthesized chemically, and

(2) The sequences immediately adjacent at the 5′ and 3′ ends at theinsertion site of the intron 3. These sequences are isolated from thehuman 28S rRNA gene comprising the insertion region of the intron 3perfectly conserved. The mobile intron will thus be placed in an rDNAenvironment. The exchange of strands will involve perfectly homologoussequences since any two are of human origin (donor sequences of theintegration cassette and recipient sequences of the human host cell).The sequences immediately adjacent at the 5′ and 3′ ends can beisolated, especially by cloning or PCR, or synthesized chemically.

1. Cloning by PCR of the Sequences Immediately Adjacent at the 5′ and 3′Ends at the Site of Insertion of the Intron 3 Starting from the 28SHuman rRNA Gene

Primers have been defined with the aid of the sequence of the human 28SrRNA gene included in the DNAstar donor bank (reference: HUMRGM, thereported sequence extending from the position+1 to the position+5025).The insertion site and the insertion region of the intron 3 have beenlocated (such as reported in Muscarella and Vogt, 1989, Cell, 56,443-454). The insertion site is included between the nucleotides 3742and 3743 of the human 28S rRNA gene.

A first fragment of DNA corresponding to the fragment extending from theposition 2324 to the position 3742 of the human 28S rRNA gene isgenerated by PCR. The primer corresponding to the coding strand(position 2324 to 2349) is reported in SEQ ID NO:4. It moreovercomprises a free 5′ end bearing the sites HindIII and NheI. The reverseprimer is described in SEQ ID NO:5 and comprises a 5′ end including thesites SpeI and XbaI. Amplification is carried out with the polymerase ofThermus Aquaticus (Perkin Elmer Cetus) according to the standardconditions indicated by the manufacturer. The matrix used is human DNAprepared conventionally. The amplified product is examined on agarosegel and sequenced.

The second fragment of human 28S rRNA gene is likewise isolated by PCR.It is the portion of gene extending from position 3743 to 4438. The twooligo-nucleotides employed are reported in SEQ ID NOS:6 and 7. The firstprimer comprises at its 5′ end a SpeI site while the reverse primercontains from 5′ to 3′ the EcoRI, NheI and BglII sites. Theamplification reaction is carried out starting from genomic human DNAprepared conventionally, under the standard conditions currentlyemployed by the specialist. The PCR fragment thus generated is examinedon agarose gel and sequenced.

The fragment comprising 1 kb of human sequences immediately adjacent atthe 5′ end at the insertion site of the intron 3 is digested by theenzymes HindIII and SpeI. The fragment comprising the sequencesimmediately adjacent in 3′ is digested by SpeI and EcoRI. The twofragments are then ligated in the plasmid pUC 19 (Yanisch-Perron et al.,1985, Gene, 33, 103-119) previously digested by HindIII and EcoRI. Theligation mixture is transformed in the strain Escherichia coli 5K (E.coli 5K) giving rise to the plasmid pHREI.

2. Cloning of the Intron 3 of Modified Physarum polycephalum

The intron 3 (0.94 kb) is isolated by the PCR technique. The primers arefixed starting from sequence data reported in the literature (Muscarellaet al, 1990, Mol. Cell. Biol., 10, 3386-3996; Johansen et al, 1992,Curr. Genet., 22, 297-304). The primers are described in SEQ ID NOS:8and 9. They both comprise a KpnI site in their 5′ region.

The amplification reaction is carried out starting from a genomic DNAbank of Physarum polycephalum, Carolina strain (Muscarella et Vogt,1989, Cell, 56, 443-454), applying the standard conditions known to thespecialist. After amplification, the size of the fragment obtained ischecked on agarose gel and its sequence is examined.

The PCR fragment is digested by KpnI and cloned in the KpnI site of thevector pUC19 to give pINEI.

The vector pINEI is digested by NheI, a unique site situated in theintron 3 of Physarum polycephalum. The linearized pINEI vector istreated with Mung bean nuclease and ligated to an XhoI linker(Stratagene). The vector resulting from this is designated pINEII andthus comprises a unique XhoI site for the insertion of a fragment of DNAof interest.

3. Insertion of the Intron 3 of Physarum polycephalum (XhoI) into aHuman 28S rRNA Gene Environment

The fragment KpnI obtained by digestion of pINEII is treated with the T4polymerase to give a fragment with three ends whose 5′ and 3′ endscorrespond exactly to those of the intron.

In parallel, pHREI is digested by the enzymes XbaI and SpeI, thentreated with the Mung bean nuclease (according to the technique reportedin Sambrook et al, 1989, Cold Spring Harbor Press). This generates alarge fragment comprising the majority of the vector pHREI, of which thefree ends correspond respectively to the nucleotides in position 3742and 3743 of the human 28S rRNA gene.

The fragment formed from pINEII comprising the intron 3 of Physarumpolycephalum is ligated to the vector pHREI treated as indicated aboveto give the vector pHREII. After introduction of a fragment of DNA ofinterest, the vector pHREI will thus comprise a transposition assemblyallowing the insertion of an integration cassette (the intron 3comprising a fragment of DNA of interest) into the human 28S rRNA gene.

Example 2 Construction of an Adenoviral Vector for the Integration ofthe Neomycin Gene into the Human Genome

An adenoviral vector is constructed comprising:

(1) the intron 3 of Physarum polycephalum (Xho⁺) at the level of whichis introduced a first expression cassette allowing the expression of theneo gene,

(2) a second expression cassette, that of the site-specific endonucleaseIPpo, encoded in part by the intron 3. The second expression cassettewill allow the production of the endonuclease IPpo for a time which issufficiently long to allow the transposition of the integration cassetteinto the genome of the host cell. The initiator codon of the translationof IPpo is situated in the exon sequence at the 5′ end of the intron 3in the 26S rRNA gene of Physarum polycephalum. The sequence immediatelyadjacent at the 5′ end to the isolated insertion site of the human 28SrRNA gene does not contain the ATG initiator present in the equivalentregion of Physarum and

(3) the appropriate elements for the maintenance of the vector in thehost cell for a time which is sufficiently long to allow thetransposition of the integration cassette.

1. Construction of the Expression Cassette of IPpo

A fragment of 167 nucleotides containing the poly A region of SV40(Fitzgerald and Shenk, 1981, Cell, 24, 251-260) is synthesized in theform of a HindIII-EcoRI fragment with the aid of an automaticsynthesizer (Milligen 7500). A BglII site is introduced just upstream ofthe EcoRI site.

A HindIII-BamHI fragment of pMSG-CAT (Pharmacia) comprising the earlypromoter of the SV40 virus is isolated. The HindIII-EcoRI syntheticfragment and the HindIII-BamHI fragment are introduced into a derivativeof the vector pUC19 (HindIII⁰) digested by BamHI and EcoRI. Thederivative is obtained after digestion of

A fragment of DNA comprising the sequence coding fro the nuclease IPpois isolated in the form of a PCR fragment. The sequence reported in theprior art (Muscarella et al., 1990, Mol. Cell. Biol., 10, 3386-3396) hasallowed two primers to be defined which are respectively described inSEQ ID NOS: 10 and 11. The 2 oligo-nucleotides comprise a HindIII siteat their 5′ end. Amplification is carried out under the standardconditions known to the specialist starting from a genomic DNA bank ofPhysarum polycephalum, Carolina strain.

The PCR fragment generated, after examination on agarose gel andsequencing, is digested by HindIII and introduced into the uniqueHindIII site of the vector pSV40EI. A clone having a correct orientationof the sequence coding for IPpo with respect to the promoter SV40 isidentified. The clone is designated pSV40EII.

2. Cloning of the Expression Cassette of the Endonuclease IPpo in theVector pHREII

The vector pHREII is digested by BglII and treated with T4 polymerase.The vector pSV40EII is digested by the enzymes PstI and BglII. Thefragment PstI-BglII comprising the expression cassette of IPpo, istreated with the Mung bean nuclease. The fragment thus treated is thenligated to the vector pHREII giving rise to pHREIII.

3. Introduction of the Expression Cassette of a DNA Fragment of Interestinto the Intron 3.

The first expression cassette of the fragment of DNA of interestcomprises in sequence from 5′ to 3′ respectively:

(1) the TK promoter of the HSV virus,

(2) the neomycin gene, and

(3) the poly A region of the SV40 virus.

The XhoI-SalI fragment of 1.1 kb comprising the said expression cassetteis isolated from the plasmid pMC1 neo (stratagene) is cloned in thevector pHREIII linearized by XhoI. The vector resulting from this isdesignated pHRE IV.

4. Cloning in an Adenoviral Vector and Infection of Host Cells

The NheI fragment comprising the transposition assembly (intron 3 intowhich is introduced the first neo expression cassette, flanked byadjacent 5′ and 3′ sequences of the human 28S RNA gene) and the secondexpression cassette of IPpo are isolated from the pHREIV vector. Thepurified fragment is treated with T4 polymerase before being insertedinto the vector pXCX2 (Spessot et al., 1989, Virology, 168, 378-387).

The vector resulting from this is used to generate a recombinantadenovirus by the conventional methods, such as, for example, the methodreported in Spessot et al. (1989, Virology, 168, 378-387). Therecombinant adenoviral vector is constructed starting from a deletionmutant of the type 5 adenovirus (Thimmappaye et al. 1982, Cell, 31,543-551). The recombinant adenoviral vector thus obtained is used toinfect human cells in culture.

The cells are cultivated under the conventional conditions known to theperson skilled in the art. 48 hours after infection, the cells areplaced in a culture medium containing neomycin.

The presence of the neo gene in the cells can be checked by theirresistance phenotype to neomycin and examined by Southern blot. Thesite-specific insertion of the integration cassette into the cellulargenome can be examined by PCR. A primer complementary to a sequence ofthe human 28S rRNA gene which is not present in the transpositionassembly (at the 5′ end of the position 2324 or at the 3′ end of theposition 4438) and a primer complementary to a sequence of theintegration cassette normally not present in the cellular genome (forexample of intron 3 or of the expression cassette of the neo gene) willpreferably be used. A PCR fragment of a uniquely defined size will beobtained in the case of site-specific integration of the integrationcassette into the human 28S rRNA gene.

Example 3 Construction of a Replicative (Episomal) Plasmid Vector forIntegration of the Neomycin Gene into the Human Genome

The NheI fragment of Example 2.4 is inserted into the pREP4 vector(Grager et al., 1989, Gene, 81, 385-294; Invitrogen Corporation, BritishBio-Technology Products LTD, Oxon, UK; reference V 004-50) at the levelof the unique NheI site.

The vector resulting from this is transfected into a line of human cellsin culture as indicated in Yates et al. (1985, Nature, 313, 812-815). Inan indicative capacity, the line 293 is mentioned, embryonic humankidney line, available from ATCC. 48 hours after transfection, the cellsare placed in a selective culture medium. The presence of the neo geneintegrated into the cellular genome is verified as described in Example2.4.

11 25 base pairs nucleic acid single linear DNA (genomic) NO syntheticoligonucleotide 1 CTATGACTCT CTTAAGGTAG CCAAA 25 12 base pairs nucleicacid single linear DNA (genomic) synthetic oligonucleotide 2 CTATGACTCTCT 12 13 base pairs nucleic acid single linear DNA (genomic) syntheticoligonucleotide 3 TAAGGTAGCC AAA 13 44 base pairs nucleic acid singlelinear DNA (genomic) NO NO synthetic oligonucleotide 4 GGGAAAAGCTTGCTAGCGGA TCTTGGTGGT AGTAGCAAAT ATTC 44 42 base pairs nucleic acidsingle linear DNA (genomic) NO YES synthetic oligonucleotide 5CCCCAAACTA GTTCTAGAGA GAGTCATAGT TACTCCCGCC GT 42 37 base pairs nucleicacid single linear DNA (genomic) NO NO synthetic oligonucleotide 6CCCCAAACTA GTAAGGTAGC CAAATGCCTC GTCATCT 37 48 base pairs nucleic acidsingle linear DNA (genomic) NO YES synthetic oligonucleotide 7AAAAGGGAAT TCGCTAGCAG ATCTCTGCTT CACAATGATA GGAAGAGC 48 33 base pairsnucleic acid single linear DNA (genomic) NO NO synthetic oligonucleotide8 TTTGGGGTAC CCACCCCCTT AAATATGGCG CTC 33 31 base pairs nucleic acidsingle linear DNA (genomic) NO YES synthetic oligonucleotide 9TTTGGGGTAC CGCTGATTCC AAACTCGGGT G 31 30 base pairs nucleic acid singlelinear DNA (genomic) NO NO synthetic oligonucleotide 10 CCCCAAAGCTTAAACAAACC ACCGCATGGA 30 34 base pairs nucleic acid single linear DNA(genomic) NO YES synthetic oligonucleotide 11 CCCAAAAGCT TCAGTGCTCTGGATGTTAAA ATGG 34

What is claimed is:
 1. A delivery vehicle including a transpositionassembly for the site-specific transfer of a DNA fragment of interestinto the genome of a mammalian host cell, said transposition assemblycomprising an integration cassette which is essentially formed by amobile genetic element in the midst of which is inserted said DNAfragment of interest; wherein (i) said mobile genetic element is devoidof a long terminal repeat and comprises an open reading frame coding foran integrase and (ii) said integration cassette is capable of beingintegrated to an insertion site specific to said genetic mobile element,said insertion site being a target sequence situated in the ribosomalnuclear DNA of said host cell, and wherein said transposition assemblyis: a) encapsulated into a liposome or synthetic lipid type vector, orb) cloned into an expression vector.
 2. The delivery vehicle of claim 1,wherein the mobile genetic element is selected from the group consistingof the mobile introns of class I, the mobile introns of class II, theretroposons of class R1, the retroposons of class R2 and the retroposonsof class R3.
 3. The delivery vehicle of claim 2, wherein the mobileintrons of class I are selected from the group consisting of: the intron3 of Physarum polycephalum, Carolina strain; the mobile intron of classI of Tetrahymena; and the mobile intron of class I of Pneumocystiscarinii.
 4. The delivery vehicle of claim 2, wherein the retroposon ofclass R2 is the retroposon of R2Bm of Bombyx mori.
 5. The deliveryvehicle of claim 2, wherein the retroposon of class R3 is the retroposonR3 of Scaria coprophila.
 6. The delivery vehicle of claim 1, whereinsaid integrase is an endonuclease.
 7. The delivery vehicle of claim 6,wherein the DNA fragment of interest is inserted into said open readingframe.
 8. The delivery vehicle of claim 1, wherein the DNA fragment ofinterest is transcribed to RNA to (i) produce an anti-sense RNA or (ii)produce a protein of interest after translation of said RNA.
 9. Thedelivery vehicle of claim 8, wherein the DNA fragment of interest isunder the control of transcription regulation elements allowing theexpression of said DNA fragment in the host cell.
 10. The deliveryvehicle of claim 9, wherein the transcription regulation elementscomprise a promoter selected from the group consisting of promotersrecognized by RNA polymerase I and by RNA polymerase II.
 11. Thedelivery vehicle of claim 1, wherein said transposition assemblycomprises, at the 5′ end of the integration cassette a region identicalto the sequences positioned at the 5′ end of the said insertion site ofthe ribosomal nuclear DNA of the host cell; and at the 3′ end of theintegration cassette a region identical to the sequences positioned atthe 3′ end of the said insertion site of the ribosomal nuclear DNA ofthe host cell.
 12. The delivery vehicle of claim 1, wherein the vectorcontains an expression cassette allowing the expression of a selectionmarker.